1. Field of the Invention
The present invention relates to a batroxobin-encoding nucleotide sequence and/or a mutated α-factor secretion signal sequence containing a specific sequence, and a vector and a transformant using the same.
2. Background of Technique
Generally, venom effect on blood coagulation cascade and fibrinolytic pathway of mammalian including human has been investigated for a long time and several effective agents have been isolated and characterized. Various components included in venom are known to affect fibrin-clotting, platelet aggregation and so on directly or indirectly, thus to be used as pro-coagulant or anti-coagulant (Meaume, J. Toxicon, 4: 2558 (1966); Matsui et al., Biochim. Biophys. Acta., 1477: 146-156 (2000)). Some of the components are already characterized and broadly used for diagnosis and therapy of thrombosis. In particular, the study about thrombin-like enzyme converting fibrinogen into fibrin by cleavage of fibrinopeptide has been performed actively and over 20 proteins have been reported, and cDNA of some was characterized.
The thrombin-like enzyme initially hydrolyze fibrinopeptide A of fibrinogen molecule to make unstable fibrin clot (des-A-fibrin) unlike thrombin, mammalian native blood coagulation protein, but the unstable fibrin clot is rapidly degraded by in vivo fibrinolysis system over time to eventually decrease blood fibrinogen level (Pirkle, H., and Stocker, K. Thromb. Haemost., 65: 444-450 (1991); Marsh, N. A., Blood Coagul. Fibrinolysis, 5: 339-410 (1994)).
Therefore, the thrombin-like enzyme is used in clinic field as hemostatic agent or therapeutic and preventing agent for thrombosis by using these both-sided characteristics of enzyme. This enzyme don't also have an influence on other blood coagulation factors and activation of platelet, with which merit it shows effective hemostatic activity to intravenously or intramuscularly inject the small amount of the enzyme (2 NIH unit/60 kg) 1-2 hrs before surgery. On the other hand, it is possible to reduce blood fibrinogen level without side effects such as bleeding, that can be happen when using thrombolytic enzyme, by controlling dose and administration time of enzyme. The release of des-A-fibrin and FDP (fibrinogen degradation products) formed during the above process stimulate heoendothelial cell to induce the production of plasminogen activator. The enzyme is used as therapeutic and preventing agent for thrombosis because the enzyme can inhibit thrombin activity (Schumacher et al., Thromb. Res., 81: 187-194 (1996); and Bell W. R. Jr., Drugs, 54: 18-30 (1997)).
Recently, this fibrinogen reduction effect of the thrombin-like enzyme is reported to be feective on therapy of heparin-induced thrombocytopenia or acute ischemic stroke caused by administration of heparin (Dempfle et al., Blood, 96: 2793-2802 (2000)).
The clinically used all thrombin-like enzymes are native proteins isolated and purified from venom. Batroxobin isolated from venom of Latin venomous snake Bothrops atrox moojeni is commercially available from Italian Solco Basle Ltd. Company and Swiss Pentapharm Company and is sold as trade names like reptilase (for hemostasis), defibrase (fro thrombolysis), reptilase-reagent (for diagnosis reagent). Botropase (for hemostasis, Italian Ravizza Company) isolated from venom of Latin venomous snake Bothrops jararaca, Malayan pit viper and Ancrod (American Knoll Pharmaceutical Company) isolated from venom of Calloselasma rhodostoma are also commercially available.
Recently, Vivostat System (Denmark, Vivosolution Co.) using batroxobin as an autologous fibrin sealant with the purpose of bleeding prevention and suture in surgical operation also is in the limelight.
Likewise, the method to produce recombinant protein has been intensively studied by various researchers since massive production batroxobin purified from snakes was limited. In the investigation to express protein from eukaryote in microorganisms, protein expression is reduced due to gene codons with low translation efficiency of E. coli (prokaryote) in the translation after transcription of eukaryotic genes. To overcome it, the method to improve protein translation is commonly carried out by using a recombinant E. coli strain into which foreign eukaryotic tRNA gene is transformed to recognize amino acid codon with low frequency in E. coli. Notwithstanding these efforts, inactive proteins are produced during refolding process of proteins expressed in E. coli (Yang et al., Biotechnol. Lett., 25: 101-104 (2003); Fan et al., Biochem. Mol. Biol. Int., 47: 217-225 (1999); Maeda et al., J. Biochem., 109: 632-637 (1991)). As reported until now, any successful case in which recombinant thrombin-like enzyme is expressed in E. coli and then has similar activity compared with specific activity of nature enzyme is not reported yet.
Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.